Diabetes is a disease characterized by above normal blood glucose levels. Diabetes occurs when the pancreas does not produce sufficient insulin or when the body becomes resistant to the effects of insulin. In both cases, the result is that glucose does not enter into the cells but instead accumulates in blood.
Insulin is a hormone which plays an important role in carbohydrate metabolism. It is released by the pancreas, for balancing glucose concentration in the blood stream after food intake. However, certain pathologies result in an insufficient or even null release of this polypeptide by the pancreas, and thus those suffering from certain diseases must be administered different amounts of this hormone.
Insulin analogues are substances obtained by modifying the structure of native insulin, which leads to significant variations in its mode of action. Structural modifications have led, for example, to analogues having a different solubility from that of native insulin, thereby controlling—upon injection—the release rate of the hormone into the blood stream. This allows for accelerating or retarding its biological effect, to meet particular patient needs.
Currently, there are two commercially available prolonged action analogues: Insulin glargine and Detemir insulin. These analogues show a longer duration of action (20-30 hours for Glargine and 20-22 hours for Detemir) with no peaks of action, as compared to NPH insulin. These pharmacokinetic differences seem to translate into improvements in adminstration regimen (every 24 hours for Glargine or 1-2 times a day, depending on patient's needs, for Detemir) and into more homogeneous plasma levels and a theoretical decrease in hypoglycemia (specially in nocturnal hypoglycemias).
Likewise, currently there are three commercially available rapid-acting analogues: Lispro, Aspart and Glulysine insulins. These three analogues have similar pharmacokinetic profiles, but which differ from that of rapid human insulin. They have a faster onset and shorter duration of action, which simulates better the endogenous response of prandial insulin. These characteristics facilitate their administration immediately before or even after food intake, thereby dispensing with the recommended waiting period of 15-30 minutes after administration of regular insulin. Various scientific publications have confirmed that the use of rapid-acting insulin provides a better glucose control than common insulin, which contributes to a better quality of life for diabetic patients.
“Rapid-acting” derivatives may be obtained by modifying certain amino acids of the insulin B chain, as disclosed, for example, in U.S. Pat. No. 5,618,913. Said document describes human insulin analogues in which an amino acid at positions B9, B12, B27 and B28 was substituted, and in which said change was enough for reducing self-association and obtaining a faster hormone action upon administration.
Further, U.S. Pat. No. 6,521,738 describes a broad variety of insulin and insulin analogue precursors, all of which contain various mini-C peptides linking insulin B and A chains. Said peptides contain at least one aromatic amino acid, a cleavage site for proteases, represented by the amino acids lysine or arginine, wherein the peptidic bond between the A chain and the peptide linker is broken, and an aromatic residue located immediately N terminal to the protease cleavage site.
U.S. Pat. No. 7,378,390 discloses examples of precursors of the des-B30 type in which proline B28 has been replaced by aspart acid. The precursors disclosed include a glycine amino acid in the peptide linker, flanked by a basic amino acid selected from Lysine or Arginine.
As disclosed in the above-mentioned patents, insulin precursors or insulin analogue precursors either lack the threonine amino acid present at position B30, designating these precursor molecules mini-C-insulin des-B30 or des-B30, or this amino acid has been replaced by another. Accordingly, in these cases, the corresponding residue must be added by means of a chemical process designated transpeptidation.
Generally, production of human insulin using recombinant DNA techniques is carried out by expressing a pro-insulin-like precursor, in which the B and A chains are linked together by a complete C chain, thus creating molecules of the insulin mini-C type where insulin B and A chains are linked together by peptides of varying sizes and sequences.
The most frequently used expression systems for producing insulin and human insulin analogues, include the microorganisms Escherichia coli and Saccharomyces cerevisiae. In the first case, they may be produced as a cytoplasmatic protein (Frank et al., 1981, in Peptides: Proceedings of the 7th American Peptide Chemistry Symposium, Rich & Gross, eds., Pierce Chemical Co., Rockford, Ill., pp. 729-739), or fused to a signal peptide to allow for secretion into the periplasmatic space (Chan et al., PNAS, 1981; 78:5401-5404). When expression is through inclusion bodies, high concentrations of the aberrantly folded, recombinant protein are obtained by means of disulphide bridge-type and hydrogen bridge bonding, causing interactions among the molecules. In order to obtain a biologically active product, the bacteria must be disrupted and inclusion bodies must be separated and diluted to isolate the peptide of interest. The latter is then refolded in vitro until its native structure is attained.
Although the use of Saccharomyces cerevisiae (Thim, L. et al., Proc. Natl. Acad. Sci., USA, 1986; 83:6766-6770; U.S. Pat. No. 4,916,212; U.S. Pat. No. 6,190,883) as a host for expressing insulin precursors or insulin analogues does not yield expression levels as high as those achieved in bacteria, it does not require renaturation steps because yeasts may carry out protein secretion into the extracellular space, thus producing the peptide with the corresponding secondary conformation. The use of this expression system comprises: fermenting yeasts so as to secrete the precursor into the culture medium; capturing the product from the supernatant, transforming the precursor into a mature product, and, lastly, final purification. During the transition from the endoplasmic reticulum to the Golgi apparatus, disulphide bonds are produced which will lead to insulin molecules, or their analogues, having properly formed disulphide bridges.
However, despite the fact that the use of S. Cerevisiae has the advantage of its simple purification process and that in vitro folding of molecules is unnecessary, it also has the disadvantage, as compared to E. Coli, of having low expression levels and, consequently, low insulin yields.
The use of methylotrophic yeasts as expression systems for recombinant proteins presents important benefits when it is necessary to produce large masses of product requiring high fermentation volumes (Cregg, J. M. et al., Mol. Cell Biol., 9:1316-1323, 1989; Cregg, J. M. et al., Bio/Technology 11:905-910, 1993).
The use of this system allows for high cell density cultures in a certain minimum medium salt solution (Brierley, R. A. et al., Ann NY Acad Sci 1990; 589:350-362), having efficient post-translational modifications (Digan, M. E., et al., in: Pierce G, ed., Development in industrial microbiology, 1988; Vol 29, Amsterdam: Elsevier Science, P59-65), low secretion of endogenous proteins during expression and secretion of the protein of interest, and further with a strong, methanol-inducible promoter, capable of being accurately regulated.
U.S. Pat. No. 7,091,032, by the same inventors as the present application, discloses the use of the above-mentioned yeasts in the manufacture of human insulin, which is hereby incorporated by reference in its entirety.
The most successful currently marketed insulin analogues are Insulin glargine (LANTUS) and aspart insulin (insulin Aspart). The former is a delayed effect analogue that achieves its effect through the addition of two basic amino acids at the carboxyl terminus of the B chain. These modifications lead to a shift of the isoelectric point of insulin, from 5.4 to 7, which will cause a decrease of solubility at physiological pH, thereby extending its absorption from the injection site.
This insulin analogue is produced in E. coli. Therefore, as mentioned previously, its precursor must be properly renaturalized, forming again the corresponding disulphide bridges.
The second of the above-mentioned analogues, aspart insulin, is obtained in the yeast Saccharomyces cerevisiae, by expression of a precursor of the miniC-desB30 type. In this case, digestion and transpeptidation reactions must be carried out in order to add the amino acid Threonine at position B30.
As described in previous publications by the authors of the present invention, the use of methylotrophic yeasts, specially those belonging to the Pichia pastoris genus, for producing insulin and insulin analogues on an industrial scale, has allowed for substantially higher yields than those achieved with the use of any of the above-mentioned microbiological expression systems.
In this sense, U.S. Pat. No. 7,091,032, by the same authors of the present application, teaches that if mini-C-type precursors are employed in which the B chain has the complete amino acid sequence, it is not necessary to carry out the transpeptidation process used in any of the previously-mentioned conventional methods.